Identification and characterization of in vivo attenuated mutants of Brucella melitensis

Brucella melitensis 16M is a Gram-negative alpha (2)-proteobacterium respon sible for abortion in goats and for Malta fever in humans. This facultative intracellular pathogen invades into and survives within both professional and non-professional phagocytes. Signature-tagged mutagenesis (STM) was use d to identify genes required for the in vivo pathogenesis of Brucella. A li brary of transposon mutants was screened in a murine infection model. Out o f 672 mutants screened, 20 were not recovered after a 5 day passage in BALB /c mice. The attenuation of 18 mutants was confirmed using an in vivo compe tition assay against the wild-type strain. The 18 mutants were characterize d further for their ability to replicate in murine macrophages and in HeLa cells. The sequences disrupted by the transposon in the mutants have homolo gy to genes coding for proteins of different functional classes: transport, amino acid and DNA metabolism, transcriptional regulation, peptidoglycan s ynthesis, a chaperone-like protein and proteins of unknown function. The mu tants selected in this study provide new insights into the molecular basis of Brucella virulence.